19-nor-cholane steroids as neurochemical initiators of change in human hypothalamic function

ABSTRACT

The invention relates to a method of altering hypothalamic function in an individual. The method comprises nasally administering a human vomeropherin, e.g. a 19-nor cholane steroid, or a pharmaceutical composition containing a vomeropherin, such that the vomeropherin binds to a specific neuroepithelial receptor. The steroid or steroids is/are preferably administered in the form of a pharmaceutical composition containing one or more pharmaceutically acceptable carriers. Other embodiments of the invention include pharmaceutical compositions containing the steroids.

This application is a divisional of application Ser. No. 08/660,804filed Jun. 7, 1996, now U.S. Pat. No. 5,922,699.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is related to U.S. application Ser. No. 08/127,908,filed Sep. 28, 1993 which is a continuation-in-part of U.S. applicationSer. No. 07/903,604, filed Jun. 24, 1992, which in turn is acontinuation-in-part of U.S. application Ser. No. 07/708,936, filed May31, 1991, which in turn is a continuation-in-part of U.S. applicationSer. No. 07/638,185, filed Jan. 7, 1991, now abandoned.

The application also relates to U.S. application Ser. No. 08/127,980filed Sep. 28,1993 which is another continuation-in-part of U.S. patentapplication Ser. No. 07/903,604, U.S. patent application Ser. No.08/077,359, filed Jun. 15, 1993, and to commonly assigned, co-pendingU.S. patent application Ser. No. 07/903,525, filed Jun. 24, 1992 (acontinuation-in-part of U.S. application Ser. No. 07/707,862, filed May31, 1991, which in turn is a continuation in-part of U.S. applicationSer. No. 07/638,743, filed Jan. 7, 1991, now abandoned) entitled“Estrene Steroids as Neurochemical Initiators of Change in HumanHypothalamic Function and Related Pharmaceutical Compositions andMethods”; and to the commonly assigned, co-pending continuation-in-partof 07/903,525, U.S. patent application Ser. No. 08/077,140. Theaforementioned U.S. patent applications are each incorporated herein byreference.

Finally, this application may relate to U.S. Patent Application entitled“Fragrance Compositions Containing Human Pheromones, filed Mar. 24,1992, U.S. Ser. No. 07/856,435.

TECHNICAL FIELD

This invention relates generally to pharmaceutical compositions andmethods for effectuating change in human hypothalamic function, therebyaltering certain behavior and physiology mediated by the hypothalamus ofindividuals. More particularly, the invention relates to the use of19-nor-cholane steroids as neurochemical effectuators of physiology andbehavior.

DESCRIPTION OF THE RELATED ART

The present invention relates to certain compounds, namely19-nor-cholane steroids, particularly 19-nor-cholane steroids andrelated compounds as will be described herein, and methods of usingthese compounds as human vomeropherins in order to alter hypothalamicfunction, thereby affecting certain consequent behavior and physiology,e.g., the reduction of anxiety. The 19-nor-cholane steroids arecharacterized by a four ring steroidal structure, methylation at the 13position and alkylation (C4) at the 17-position. The 19-nor-cholenes area subset which have at least one double bond.

Ohloff, G. et al. (Helv. Chim. Acta (1983) 66:192-217), which isincorporated herein by reference, have shown that several steroids(androstenes) have an odor which varies with different isomeric,diastereomeric, and enantiomeric forms. Some members of this group havebeen reported to act as a pheromone in some mammalian species forinstance, 5α-androst-16-en-3-one and 5α-androst-16-en-3α-ol in pigs(Melrose, D. R., et al., Br. vet. J. (1971) 127:497-502). These16-androstenes produced by the boar induce mating behavior in estrussows (Claus, et al., Experimentia (1979) 35:1674-1675).

In some studies it has been noted that, in some species, variouscharacteristics of certain 16-androstenes (including5α-Androst-16-en-3α-ol and 5α-Androst-16-en-3-one), such asconcentration, metabolism, and localization, are sexually dimorphic(Brooksbank et al., J. Endocr. (1972) 52:239-251; Claus, et al., J.Endocr. (1976) 68:483-484; Rwan, et al., Med. Sci. Res. (1987)15:1443-1444). For instance, 5α-Androst-16-en-3α-ol and5α-Androst-16-en-3-one, as well as Androsta-4,16-dien-3-one, have beenfound at different concentrations in the peripheral blood, saliva andaxillary secretions of men and of women (Kwan, T. X., et al., Med. Sci.Res. (1987) 15:1443-1444), and their function as a human pheromone, tothe extent of affecting choice and judgement, has been suggested (Id.;see also Gower, et al., “The Significance of Odorous Steroids inAxillary Odour”, In, Perfumery, pp. 68-72, Van Toller and Dodd, Eds.,Chapman and Hall, 1988); Kirk-Smith, D. A., et al., Res. Comm. Psychol.Psychiat. Behav. (1978) 3:379). Androstenol (5α-androst-16-en-3α-ol) hasbeen claimed to exhibit a pheromone-like activity in a commercial men'scologne and women's perfume (Andron for men and Andron for women byJovan). Japanese Kokai No. 2295916, refers to perfume compositionscontaining androstenol and/or its analogues. 5α-Androstadien-3β-ol (andperhaps the 3α-ol) has also been identified in human axillary secretion(Gower, et al., Supra at 57-60. On the other hand, there is littleagreement in the literature as to whether or not any putative pheromoneactually plays any role in the sexual or reproductive behavior ofmammals, particularly of humans. See: Beauchamp, G. X., et al., “ThePheromone Concept in Mammalian Chemical Communication: A Critique”, In:Mammalian Olfaction. Reproductive Processes and Behavior, Doty R. L.,Ed., Academic Press, 1976). See also: Gower, et al., supra at 68-73.

The pheromone properties of some estrene steroids for some mammalianspecies have been described. Michael, R. P. et al., Nature (1968)218:746 refers to Estrogens (particularly Estradiol) as a pheromonalattractant of male rhesus monkeys. Parrot, R. F., Hormones and Behavior(1976) 7:207-215, reports Estradiol benzoate injection induces matingbehavior in ovariectomized rats; and the role of the blood level ofEstradiol in make sexual response (Phoenix, C. H., Physiol. and Behavior(1976) 16:305-310) and female sexual response (Phoenix, C. H., Hormonesand Behavior (1977) 8:356-362) in Rhesus monkeys has been described. Onthe other hand, there is little agreement in the literature as towhether or not pheromones as such play any role in the reproductivebehavior and interpersonal communication of mammals (Beuchamp, G. K., etal., “The Pheromone Concept in Mammalian Chemical Communication: ACritique”, In: Mammalian Olfaction Reproductive Processes. and Behavior,Doty, R. L., Ed., Academic Press, 1976).

An embodiment of the subject invention concerns the non-systemic, nasaladministration of certain 19-nor-cholane and 19-nor-cholene steroids toaffect a specific behavioral or physiological response in humansubjects, e.g., a reduction of negative affect, mood, and charactertraits. In particular, nasal administration provides for contactingneurochemical receptors of a heretofore poorly understood neuroendocrinestructure, commonly known as the vomeronasal organ (“VNO); also known as“Jacobson's organ”), with one or more steroid(s) or with compositionscontaining the steroid(s). This organ is accessed through the nostrilsof most higher animals—from snakes to humans, and has been associated,inter alia, with pheromone reception in certain species (see generallyMuller-Schwarze & Silverstein, Chemical Signals, Plenum Press, New York(1980)). The axons of the neuroepithelia of the vomeronasal organ,located supra palatinal, form the vomeronasal nerve and have directsynaptic connection to the accessory olfactory bulb and indirect inputfrom there to the cortico-medial amygdaloid basal forebrain andhypothalamic nuclei of the brain. The distal axons of terminalis nerveneurons may also serve as neurochemical receptors in the VNO. Stensaas,L. J., et al., J. Steroid Biochem. and Molec. Biol. (1991) 39:553. Thisnerve has direct synaptic connection with the hypothalamus.

Johnson, A. et al. (J. Otolarynaoloav (1985) 14:71-79) report evidencefor the presence of the vomeronasal organ in most adult humans, butconclude that the organ is probably non-functional. Contravening resultswhich suggest that the VNO is a functional chemosensory receptor arereported by Stensaas, L., et al., supra; and by Moran, D. T., et al.,Garcia-Velasco, J. and M. Mondragon; MontiBloch, L. and B. Grosser allin J. Steroid Biochem. and Molec. Biol. (1991) 39.

It is apparent that it would be desirable to identify and synthesizehuman vomeropherins and pheromones and to develop pharmaceuticalcompositions and methods of use to influence hypothalamic function. Thisinvention relates to the unexpected discovery that, when nasallyadministered to human subjects, certain neurochemical ligands,particularly 19-nor-cholane steroids, 19-nor-cholene steroids andrelated compounds, or pharmaceutical compositions containing19-nor-cholanes, 19-nor-cholenes or related compounds, specifically bindto chemoreceptors of certain nasal neuroepithelial cells and thisbinding generates a series of neurophysiological responses resulting inan alteration of hypothalamic function of an individual. When properlyadministered, the effect of certain of these compounds on thehypothalamus affects the function of the autonomic nervous system and avariety of behavioral-or physiological phenomena which include, but arenot limited to the following: anxiety, premenstrual stress, fear,aggression, hunger, blood pressure, and other behavioral andphysiological functions normally regulated by the hypothalamus. See OttoAppenzeller, The Autonomic Nervous System. An Introduction of Basic andClinical Concepts (1990); Rorner, P. I. Central nervous control ofautonomic cardiovascular function, and Levy N. M. and Martin, P. J.Neural control of the heart, both in Handbook of Physiology: Section2:Cardiovascular System—the heart, Vol. I, Washington, D.C., 1979,American Physiological society; Fishman, A. P., et al. editors, Handbookof Physiology: Section3: Respiratory System. Vol. II. Control ofBreathing, Bethesda Md. 1986. American Physiological Society.

In some instances a single 19-nor-cholane steroid, or related compound,is administered, in some instances combinations of 19-nor-cholanesteroids and/or related compounds are administered and in some instancesone or more 19-nor-cholane steroids are coadministered along with one ormore estrane or estrene steroids, androstane or androstene steroids or arelated compound.

BACKGROUND OF THE INVENTION Definitions

An “affect” is a transient feeling state. Typical negative affects arefeelings of nervousness, tenseness, shame, anxiousness, irritability,anger, rage, and the like. “Moods” are longer lasting feeling statessuch as guilt, sadness, hopelessness, worthlessness, remorsefulness,misery, unhappiness and the like. “Character traits” are more permanentaspects of an individual's personality. Typical negative charactertraits are sensitivity, regretfulness, blameworthiness, stubbornness,resentfulness, bitterness, timidness, laziness and the like.

“19-nor-cholane steroids” are aliphatic polycyclic hydrocarbonscharacterized by a four-ring steroidal structure with a methylation atthe 13-position and alkylation (C4 or higher) (including unsaturatedgroups) at the 17-position. The 19-nor compounds lack a methyl or othercarbon-containing substituent on C-10 where C-19 would normally befound. A cholene is a subset of cholanes commonly understood to meanthat the compound has at least one double bond.

A “chemoreceptor” is a receptor molecule displayed on the surface of a“chemosensory” neuroepithelial cell which binds in a stereospecificfashion to a particular ligand or ligands. This specific bindinginitiates a signal transduction which initiates an afferent nerveimpulse. Chemoreceptors are found, inter alia, in taste buds, olfactoryepithelium and vomeronasal tissue.

“19-nor-cholene steroids”, as the term is used herein, are aliphaticpolycyclic hydrocarbons with a four-ring steroidal structure, at leastone double bond in the A-ring, methylation at the 13-position,alkylation (C4 or higher) (including unsaturated groups) at the17-position and an oxo, hydroxyl or hydroxyl derivative such as analkoxy, ester, benzoate, cypionate, sulfate or glucuronide, at the3-position. The 19-nor compounds lack a methyl or other carbon-containersubstituent or C-10 where C-19 would normally be found.

The following structure shows the four-ring steroidal structure commonto cholane and cholene steroids. In describing the location of groupsand substituents, the following numbering system will be employed:

“Sexually dimorphic” refers to a difference in the effect of, orresponse to, a pharmaceutical agent between males and females of thesame species.

An “effective amount” of a drug is a range of quantity and/orconcentration which brings about a desired physiological and/orpsychological effect when administered to an individual in need of thedrug. In the present case, a needy individual is one with aphysiological or behavioral trait which is normally regulated by thehypothalamus and wherein it is desirable to affect the function of thehypothalamus or the trait. The effective amount of a given drug may varydepending upon the function to be affected, the desired effect, route ofadministration, and the like. For example, when the steroid inadministered as a solution applied to the facial skin of a subject aneffective concentration is from 1 microgram/ml to 100 μg/ml, preferably10 to 50 μg/ml and most preferably 20 to 30 μg/ml. When the steroid isintroduced directly into the VNo an effective amount is about 1 picogramto about 1 nanogram, more preferably about 10 picograms to about 50picograms. When the steroid is administered to the nasal passage, byointment, cream or aerosol, or the like, an effective amount is about100 pg to about 100 micrograms, preferably about 1 ng to about 10micrograms. It follows that some drugs may be effective whenadministered by some routes, but not effective when administered byother routes.

The “hypothalamus” is the portion of the diencephalon comprising theventral wall of the third ventricle below the hypothalamic sulcus andincluding structures forming the ventricle floor, including the opticchiasma, tuber cinereum, infundibulum, and mammillary bodies. Thehypothalamus regulates the autonomic nervous system and controls severalphysiological and behavioral functions such as the so-called fight andflight responses, sexual motivation, water balance, sugar and fatmetabolism, hunger, regulation of body temperature, endocrinesecretions, and others. The hypothalamus is also the source ofvasopressin which regulates blood pressure, and oxytocin which inducesparturition and milk release. All hypothalamic functions are potentiallymodulatable by the vomeropherin therapy described herein.

A “ligand”, as used herein, is a molecule which acts as a chemicalsignal by specifically binding to a receptor molecule displayed on thesurface of a receptor cell, thereby initiating a signal transductionacross the cell surface. Binding of ligands to chemosensory receptorscan be measured. Chemosensory tissue, such as vomeronasalneuroepithelium or olfactory neuroepithelium, contains a multiplicity ofneuroreceptors cells, each displaying at least one cell surfacereceptor. Many of the receptor molecules have identical ligandspecificity. Therefore, when the tissue is exposed to a ligand for whichit has specificity (for example a exposure of the VNO to a vomeropherin)a summated change in cell surface receptor potential can be measured.

As used herein, “lower alkyl” means a branched or unbranched saturatedhydrocarbon chain of 1 to 4 carbons, such as, for example, methyl,ethyl, n-propyl, i-butyl and the like. “Alkoxy” as used herein is usedin its conventional sense to mean the group —OR wherein R is alkyl asherein defined.

A “pheromone” is a substance that provides chemical means ofcommunication between members of the same species through secretion andperipheral chemoreception. In mammals pheromones are usually detected byreceptors in the vomeronasal organ of the nose. Commonly, pheromoneseffect development, reproduction and related behaviors. A “vomeropherin”is a more general term which includes pheromones and describes asubstance from any source which functions as a chemosensory messenger,binds to a specific vomeronasal neuroepithelial receptor, and induces aphysiological or behavioral effect. The physiologic effect of a“vomeropherin” is mediated through the vomeronasal organ.

A picogram (pg) is equal to 0.001 nanograms (ng). A ng is equal to 0.001micrograms (μg). A μg is equal to 0.001 mg.

The invention is directed to a group of certain 19-nor cholane steroids.

A subset of 19-nor-cholanes within the group are believed to be novel.Syntheses are described herein for certain compounds in the Schemes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 and FIG. 2 show the EVG amplitudes of steroids E2/NC2, E1/NC2,E2/NC3, E1/NC3, methylated E2/NC2, methylated E2/NC3, E8/NC3 (Chart), inhuman male and female VNO'S, respectively.

SUMMARY OF THE INVENTION

Accordingly, it is an object of this invention to provide pharmaceuticalcompositions which contain human vomeropherins or pheromones and aresuitable for nasal administration in an individual.

It is also an object of this invention to provide methods of using thesecompositions to alter hypothalamic function of an individual.

It is a further object of this invention to provide methods of usingthese compositions to affect physiological and behavioral functions ofindividuals which are normally regulated by the hypothalamus.

Finally, it is an object of this invention to provide methods ofaltering hypothalamic function which have the following advantages: 1)administration directly to the chemoreceptors in the nasal passage andthe vomeronasal organ, without pills or needles—i.e., non-invasively; 2)a mode of drug action through the nervous system and not through thecirculatory system—thus brain function can be affected withoutconsideration of the bloodbrain barrier; 3) a direct means of affectingthe hypothalamus—there is only one synaptic junction between pheromonereceptors and the hypothalamus; and, 4) providing a highly specific drugeffect, thereby greatly reducing the potential for undesirableside-effects—this because sensory nerves are addressed to a specificlocation in the brain. Additional objects, advantages and novel featuresof the invention will be set forth in part in the description whichfollows, and in part will become apparent to those skilled in the artupon examination of the following, or may be learned by practice of theinvention.

Objects of this invention are achieved by providing a pharmaceuticalcomposition suitable for nasal administration in an individual. Thecomposition contains a pharmaceutically acceptable carrier and a cholanesteroid with the formula:

wherein P₁ is oxo, α- or β-hydroxy, α- or β-acetoxy, α- or β-propionoxy,α- or β-lower acetoxy, α- or β-lower acyloxy, or α- or β-benzyloxy;

“a”, “b”, “c”, “d”, “e”, “f”, “g”, “h”, “i”, “j”, “m”, “s” and “n” arealternative sites for optional double bonds, and “k” may be absent orpresent with “j” to form a triple bond;

P₂ is hydroxy, hydrogen, lower alkoxy of 1 to 6 carbon atoms, or P₂ isabsent;

P₃ is oxo, hydrogen, hydroxy, lower alkoxy of 1-6 carbon atoms or halo;

P₄ is methyl or ethyl;

each P₅ and P₇ independently is hydrogen, methyl or halo;

P₆ is hydrogen or methyl;

R′ and R″ are independently hydrogen or halo, are absent, or togetherform=CH₂; q is an integer from D to 2.

Preferably, q=1.

One class of preferred steroid compositions contain steroids wherein “d”is a double bond, and optionally “b” is present as a double bond.Another preferred class has “a”, “d” and “e” present, and g or h areoptionally present. If “g” is present in this case, then “n” isoptionally present. Other preferred classes have “c” or “s” present.

The novel class of 19-nor-cholanes are those of the above formula,excluding the compounds in the instances where P₃, P₆, P₅, P₇, R′ and R″are hydrogen; P₄ is methyl, e, a, d are present; b, c, f, g, h, i, j, k,n, and s are absent, q=o, P₁ is hydroxy and m is present or absent.

The term lower alkyl, lower alkoxy, etc., is meant to encompass carbonchains of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms.

Other objects of this invention are achieved by providing a method ofaltering hypothalamic function and/or autonomic function in anindividual. A ligand for a chemoreceptor displayed on the surface of anasal neuroepithelial cell is provided wherein the cell is a part oftissue other than olfactory epithelia; and, the ligand is administeredwithin a nasal passage of the individual such that the ligand bindsspecifically to the chemoreceptor, resulting in an alteration ofhypothalamic function of the individual.

All embodiments of this application relate to and include the functionalequivalents of the steroid structures disclosed in these embodiments andto those modified steroids which demonstrate said functionalequivalence,

In the following chart, particularly preferred 19-nor-cholanes areshown.

19-NORCHOLANES NC E 1 2 3 1

NOVEL NOVEL NOVEL 2

NOVEL KNOWN KNOWN 3

NOVEL NOVEL NOVEL 4

NOVEL NOVEL NOVEL 5

NOVEL NOVEL NOVEL 6

NOVEL NOVEL NOVEL 7

NOVEL NOVEL NOVEL 8

NOVEL NOVEL NOVEL 9

NOVEL NOVEL NOVEL 10

NOVEL NOVEL NOVEL 11

NOVEL NOVEL NOVEL 12

NOVEL NOVEL NOVEL 13

NOVEL NOVEL NOVEL 14

NOVEL NOVEL NOVEL NC E 4 5 1

NOVEL NOVEL 2

NOVEL NOVEL 3

NOVEL NOVEL 4

NOVEL NOVEL 5

NOVEL NOVEL 6

NOVEL NOVEL 7

NOVEL NOVEL 8

NOVEL NOVEL 9

NOVEL NOVEL 10

NOVEL NOVEL 11

NOVEL NOVEL 12

NOVEL NOVEL 13

NOVEL NOVEL 14

NOVEL NOVEL

Alkoxy derivatives are prepared from their corresponding hydroxysteroids by reaction with an alkylating agent such as trimethyloxoniumfluoroborate, triethyloxonium fluoroborate or methylfluorosulfonate inan inert chlorocarbon solvent such as methylene chloride. Alternatively,alkylating agents such as alkyl halides, alkyl tosylates, alkylmesylates and dialkylsulfate may be used with a base such as NaH, KM orKOBut, silver oxide or barium oxide in polar, aprotic solvents as forexample, DMF, DMSO and hexamethylphosphoramide.

General procedures for synthetic reactions of steroids are known tothose skilled in art. Where time and temperature of reactions must bedetermined, these can be determined by a routine methodology. Afteraddition of the required reagents, the mixture is stirred under an inertatmosphere and aliquots are removed at hourly intervals. The aliquotsare analyzed by chromatography to monitor the disappearance of startingmaterial, at which point the work-up procedure is initiated. If thestarting material is not consumed within twenty-four hours, the mixtureis heated to reflux and hourly aliquots are analyzed, as before, untilthe starting material disappears. In this case the mixture is allowed tocool before the work-up procedure is initiated.

Purification of the products is accomplished by means of chromatographyand/or crystallization, as known to those skilled in the art.

Pharmaceutical Compositions and Methods of Use

An embodiment of the subject invention is a method of altering thehypothalamic function of an individual. Another embodiment is alteringan autonomic function of an individual. These autonomic functionsinclude but are not limited to heart rate, respiratory rate, brain wavepatterns (percentage alpha cortical activity), body temperature. Otherembodiments include, but are not limited to, methods of diminishingnegative affect, negative mood or negative character traits of anindividual. Another embodiment is a method of treating femalepremenstrual stress. All of these embodiments are accomplished by meansof the non-systemic, nasal administration of certain cholane steroids,combinations of cholane steroids and combinations of one or more cholanesteroids and one or more androstane and/or estrene steroids.

This particular mode of administration is distinguished from alternativemodes, such as ingestion or injection, in several important ways, theseby virtue of the direct contact with the VNO provided by the nasaladministration of the steroid ligand. In the methods of this invention,the appropriate ligand is administered directly to the chemoreceptors inthe nasal passage and the vomeronasal organ, without pills orneedles—i.e., non-invasively. Drug action is mediated through binding ofthe ligands, described herein, to specific receptors displayed byneuroepithelial cells in the nose, preferably in the VNO. Thisfurthermore, the mode of drug action is through the nervous system andnot through the circulatory system—thus brain function can be affectedwithout consideration of the blood-brain barrier. These methods oftreatment provide a direct means of affecting the hypothalamus throughthe nervous system because there is only one synaptic junction betweenpheromone receptors and the hypothalamus. Because sensory nerves areaddressed to a specific location in the brain, this method has a highlyspecific drug effect, thereby greatly reducing the potential ofundesirable side-effects.

VNO contact is important because the VNO is associated withchemoreceptive/pheromonal function. The VNO consists of a pair of blindtubular diverticula which are found at the inferior margin of the nasalseptum. The VNO contains neuro-epithelia, the axons of which have directsynapses to the amygdala and from there, to the hypothalamus. Theexistence of the VNO has been well documented in most terrestrialvertebrates including the human fetus; however, in adult humans it isgenerally thought to be rudimentary (See Johnson, et al., supra).

Stimulation of the hypothalamus via the VNO may allow one to suppressrelease of LH and FSH. This can provide a clinical method for treatmentof prostatic cancer, precocious puberty (in males and females),endometriosis, uterine leiomyoma, breast cancer, premenstrual syndromeand dysfunctional uterine bleeding.

The ligand substances described herein, or their sulfated cypionated,benzoated, proprionated, or glucuronated derivatives, may beadministered directly, but are preferably administered as compositions.They are prepared in a liquid dosage form such as, for example, liquids,suspensions or the like, preferably in unit dosage forms suitable forsingle administration of precise dosages. Liquid dosages may beadministered as nose drops or as an aerosol. Alternatively, the activecompound can be prepared as a creme or an ointment composition andapplied topically within the nasal cavity. In addition, a vomeropherinmay be administered as vapor contained in an air puff delivered in thenasal cavity. As another alternative, delivery may occur by controlledrelease of these agents by encapsulation either in bulk or at amicroscopic level using synthetic polymers, such as silicone, andnatural polymers such as gelatin and cellulose. The release rate can becontrolled by proper choice of the polymeric system used to control thediffusion rate (Langer, R. S. and Peppas, N. A., Biomaterials 2,201,1981). Natural polymers, such as gelatin and cellulose slowly dissolvein a matter of minutes to hours while silicone remains intact for aperiod of months. The compositions will include a conventionalpharmaceutical carrier or excipient, one or more of the active19-nor-cholane compound(s), and the composition may or may notadditionally include one or more androstane or estrene steroids. Inaddition, the compositions may include other medicinal agents,pharmaceutical agents, carriers, adjuvants, etc.

The most likely means of communication of a semiochemical ligand is theinhalation of a naturally occurring pheromone present on the skin ofanother. It is estimated that the naturally occurring maximumconcentration of a cholane steroid on human skin is from 2 to 7 ng/cm².During intimate contact it is estimated that a human would be exposed tono more than 700 ng of a naturally occurring steroid. Since thesecompounds are relatively nonvolatile, it is estimated that, even duringintimate contact, a human subject would inhale no more than 0.7 pg of anaturally occurring steroid from the skin of another. From the a countinhaled only about 1% would reach the receptors of the vomeronasalorgan. Thus the estimated maximum natural exposure to naturally producedpheromones would be 0.007 pg.

The amount of vomeropherin administered will of course, be dependent onthe subject being treated, the severity of the affliction, the manner ofadministration, the frequency of administration, and the judgment of theprescribing physician. However, a single dosage of at least about 10picograms, delivered directly into the lumen of the vomeronasal organ,is effective in eliciting a transient autonomic response. Whenadministered to the nasal cavity, the dosage is about 100 picograms toabout 10 micrograms, preferably about 1 nanogram to about 10 micrograms,more preferably about 10 nanograms to 1 about microgram. The frequencyof administration is desirably in the range of an hourly dose to amonthly dose, preferably from 8 times/day to once every other day, morepreferably 1 to 3 times per day. Ointments containing one or more activecompounds and optional pharmaceutical adjuvants in a carrier, such as,for example, water, saline, aqueous dextrose,. glycerol, ethanol, andthe like, can be prepared using a base such as, for example, petroleumjelly, lard, or lanolin.

Liquified pharmaceutically administrable compositions can, for example,be prepared by dissolving, dispersing, etc. an active compound asdefined above and optional pharmaceutical adjuvants in a carrier, suchas, for example, water, saline, aqueous dextrose, glycerol, ethanol, andthe like, to thereby form a solution or suspension. If desired, thepharmaceutical composition to be administered may also contain minoramounts of nontoxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents and the like, for example, sodium acetate,sorbitan monolaurate, triethanolamine sodium acetate, triethanolamineoleate, etc. Actual methods of preparing such dosage forms are known, orwill be apparent, to those skilled in this art; for example, seeRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa,15th Ed., 1975. The composition or formulation to be administered will,in any event, contain a quantity of one or more of the activecompound(s) in an amount effective to alleviate the symptoms of thesubject being treated.

For aerosol administration, the active ingredient is preferably suppliedin finely divided form along with a surfactant and a propellant. Typicalpercentages of active ingredients are 0.001 to 2% by weight, preferably0.004 to 0.10%.

Surfactants must, of course, be nontoxic, and preferably soluble in thepropellant. Representative of such agents are the esters or partialesters of fatty acids containing from 6 to 22 carbon atoms, such ascaproic, octanoic, lauric, palmitic, stearic, linoleic, olestearic andoleic acids with an aliphatic polyhydric alcohol or its cyclic anhydridesuch as, for example, ethylene glycol, glycerol, erythritol, arabitol,mannitol, sorbitol, and hexitol anhydrides derived from sorbitol (thesorbitan esters sold under the trademark “Spans”) and thepolyoxyethylene and polyoxypropylene derivatives of these esters. Mixedesters, such as mixed or natural glycerides, may be employed. Thepreferred surface-active agents are the oleates or sorbitan, e.g., thosesold under the trademarks “Arlacel C” (sorbitan sesquioleate), “Span 80”(sorbitan monoleate) and “Span 85” (sorbitan trioleate). The surfactantmay constitute 0.1-20% by weight of the composition, preferably 0.25-5%.

The balance of the composition is ordinarily propellant. Liquifiedpropellants are typically gases at ambient conditions, and are condensedunder pressure. Among suitable liquefied propellants are the loweralkanes containing up to five carbons, such as butane and propane;fluorinated or fluorochlorinated alkanes, such as are sold under thetrademark “Freon”. Mixtures of the above may also be employed.

In producing the aerosol, a container equipped with a suitable valve isfilled with the appropriate propellant, containing the finely dividedactive ingredient and surfactant. The ingredients are thus maintained atan elevated pressure until released by action of the valve.

Yet another means of administration is topical application of a volatileliquid composition to the skin, preferably facial skin, of anindividual. The composition will usually contain an alcohol such asethanol or isopropanol. A pleasant odorant may also be included in thecomposition.

Measuring Affect, Mood and Character Trait

Feeling states associated with affects, moods and character traits aregenerally measured by use of a questionnaire. For example questionnairescomprising a number of adjectives which refer to feeling states may beadministered to an individual. The individual evaluates his or herfeeling state described by the adjective and rates the intensity of thefeeling on a numerical scale. Clustering of related adjectives andstatistical analysis of a subject's evaluation of each adjectiveprovides a basis for the measurement of various feeling states.

Alternatively, feeling states may be measured by autonomic changes, suchas those used in polygraphic evaluations (galvanic skin response, pulserate and the like). Cabanac, M. Annual Review of Physiology (1975)37:415; Hardy, J. D., “Body Temperature Regulation”, Chapter 59, pp.1417. In: Medical Physiology. Vol. II Ed.: VB Mountcastle (1980);Wolfram Bouscein. Electrodermal Activity (Plenum Press 1992). Inaddition, non-verbal cues such as facial expression and body posture maybe evaluated.

EXAMPLES

The following examples are intended to illustrate but not to limit theinvention.

Abbreviations used in the examples are as follows: aq.=aqueous; RT.=roomtemperature; PE=petroleum ether (b.p. 50-70°);DMF=N,N-dimethylformamide; DMSO=dimethyl sulfoxide; THF=tetrahydrofuran.

Example 1 19,21-Bisnorchola-1,3.5(10),17Z-tetraen-3-yl Methyl Ether, 2

To a solution of 19,21-bisnorchola-1,3,5(10),17Z-tetraen-3β-ol (1,1.0000 g, 3.2208 mmol) in 50 mL of acetone was added potassium carbonate(0.67 g, 4.8 mmol), and the resulting suspension was heated to refluxwith exclusion of moisture. Dimethyl sulfate (0.76 mL, 8.0 mmol) wasadded and reaction was continued 22 h. The mixture was then poured into50 mL of 5% (w/w) sodium hydroxide and extracted 3 times with 50 mLportions of ether. The combined organic extracts were washed 3 timeswith 50 mL portions of brine, dried over magnesium sulfate, and filteredthrough diatomaceous earth. The residue was washed with 25 mL of etherand the combined filtrates were concentrated under reduced pressure.Crystallization of the residual tan solid from 95% ethanol withintermediate treatment with charcoal yielded lustrous white platelets(753.1 mg, 2.321 mmol, 72%), m.p. 80.5-82° C., homogeneous to TLC (10%ethyl acetate/hexanes on silica gel; product R_(f)0.69;estra-1,3,5(10),16-tetraen-3-yl methyl ether R_(f)0.66).

Example 2 19,21-Bisnorchola-2.5(10),17Z-trien-3-yl Methyl Ether, 3

A solution of 19,21-bisnorchola-1,3,5(10),17Z-tetraen-3-yl methyl ether(2, 450.0 mg, 1.387 mmol) in 13 mL of anh. THF+4.60 g (62.1 mmol) oft-butanol was added to ca. 50 mL of anh. ammonia, followed by 0.20 g (29mg-atom) of lithium wire cut in small pieces. Reaction was continued for7 h, after which 1.6 mL of methanol were added and ammonia was allowedto boil off overnight. 40 mL of water were added and the mixture wasextracted 3 times with 40 mL portions of ether. The combined organicextracts were washed 3 times with 40 mL portions of brine, dried overmagnesium sulfate, and filtered through Celite 503. The residue waswashed with 10 mL of ether and the combined filtrates were concentratedunder reduced pressure. Flash filtration of the resulting whiteplatelets (hexanes—1% ethyl acetate/hexanes—2% ethyl acetatethexanes)followed by recrystallization from 95% ethanol gave lustrous, fine,white platelets (329.7 mg, 1.010 mmol, 73%), m.p. 76-77° C. TLC (1%ethyl acetate/hexanes) showed this to be a mixture of the desired Birchproduct (R_(f)0.15) and starting material (R_(f)0.09).

Example 3 19,21-Bisnorchola-4,17Z-dien-3 One, 4

To a solution of crude 19,21-bisnorchola-2,5(10),17Z-trien-3-yl methylether (3, 130 mg, 0.3981 mmol) in 35 mL of acetone were added 1.3 mL ofmethanol and 1.3 mL of con. (12.1 M) HCl. After stirring 1 h, 1.33 g ofsodium bicarbonate +10 mL of water were added and the mixture wasextracted 3 times with 5 mL portions of methylene chloride. The combinedorganic extracts were washed with 5 mL of brine, dried over sodiumsulfate, and filtered through Celite 503. The residue was washed with 5mL of methylene chloride and the combined filtrates were concentratedunder reduced pressure. Preparative TLC (10% ethyl acetatelhexanes onalumina GF, 1000 μ) of the resulting yellow syrup produced a slightlyyellow resin (293 mg, 93.7 μmol, 24%) homogeneous to TLC (10% ethylacetate/hexanes on silica gel; product R_(f)0.1; estra-4,16-dien-3-oneR_(f)0.1).

Example 4 19,21-Bisnorchola-1,3,5(10)-trien-3-yl Methyl Ether, 6

To a solution of 19,21-bisnorchola-1,3,5(10)-trien-3-ol (5, 460.0 mg,1.472 mmol) in 25 mL of acetone was added potassium carbonate (0.31 g,2.2 mmol), and the suspension was heated to reflux with exclusion ofmoisture. Dimethyl sulfate (0.34 mL, 3.6 mmol) was added and reactionwas continued for 20 h. The mixture was then poured into 25 mL of 5%(w/w) sodium hydroxide and extracted 3 times with 25 mL portions ofether. The combined organic extracts were washed 3 times with 25 mLportions of brine, dried over magnesium sulfate, and filtered throughdiatomaceous earth. The residue was washed with 10 mL of ether and thecombined filtrates were concentrated under reduced pressure. Flashchromatography (10% ethyl acetate/hexanes on silica gel) gave acolorless syrup (0.43 g, 1.3 mmol, 89%).

Example 5 19,21-Bisnorchola-2,5(10)-dien-3-yl Methyl Ether, 7

A solution of 19,21-bisnorchola-1,3,5(10)-trien-3-yl methyl ether (6,0.36 g, 1.1 mmol) in 10 mL of anh. THF +3.68 g (49.6 mmol) of t-butanolwas added to ca. 35 mL of anh. ammonia, followed by 0.16 g (23 mg atom)of lithium wire cut in small pieces. Reaction proceeded for 8 h and wasthen quenched with 1.3 mL of methanol. After allowing ammonia to boiloff overnight, 30 mL of water were added and the mixture was extracted 3times with 30 mL portions of ether. The combined organic extracts werewashed 3 times with 30 mL portions of brine, dried over magnesiumsulfate, and filtered through Celite 503. The residue was washed with 25mL of ether and the combined filtrates were concentrated under reducedpressure to give a colorless syrup (0.33 g, 1.0 mmol, 91%) homogeneousto TLC (5% ethyl acetate/hexanes on silica gel; R_(f)0.69; startingmaterial R_(f)0.56 0.49).

Example 6 19.21-Bisnorchol-4-en-3-one, 8

To a solution of 19,21-bisnorchola-2,5(10)-dien-3-yl methyl ether (7,0.27 g, 0.82 mmol) in 7.2 mL of acetone were added 2.3 mL of methanoland 2.3 mL of con. (12.1 M) HCl. After stirring 1 h, 2.75 g of sodiumbicarbonate and 20 mL of water were added and the mixture was extracted3 times with 10 mL portions of methylene chloride. The combined organicextracts were washed with 10 mL of brine, dried over sodium sulfate, andfiltered through Celite 503. The residue was washed with 10 mL ofmethylene chloride and the combined filtrates were concentrated underreduced pressure. Preparative TLC (20% ethyl acetate/hexanes on aluminaGF, 1000 μ) gave a light yellow syrup (125.6 mg, 0.3994 mmol, 49%)homogeneous to TLC (20% ethyl acetate/hexanes on silica gel; productR_(f)0.39; estra-4,16-dien-3-one R_(f)0-34).

Example 7 Electrophysiological Studies

The following electrophysiological studies were performed in clinicallynormal human volunteers of both sexes whose ages ranged from 19 to 29years. No anesthetics were used, and female subjects were excluded ifpregnant.

The stimulation and recording system consists of a “multifunctionalminiprobe” described elsewhere (Monti-Bloch, L. and Grosser, B. l.(1991) “Effect of putative pheromones on the electrical activity of thehuman vomeronasal organ and olfactory epithelium,” J. Steroid Biochem.Molec. Biol. 39:573582). The recording electrode is a 0.3 mm silver ballattached to a small (0.1 mm) silver wire insulated with Teflon© thesurface of the electrode is first treated to produce a silver chlorideinterface, and is then covered with gelatin It is positioned within asmall caliber Teflon© catheter (dia=5 mm) such that the tip of theelectrode protrudes approximately 2 mm. The Teflon© catheter is 10 cm inlength and constitutes the terminal extension for a multichanneldelivery system which delivers a continuous air stream carrying discreetpulses of chemosensory stimuli. The air stream first passes into a smallchamber and is bubbled through a solution containing either avomeropherin or an olfactant in a diluent or the diluent alone. Asolenoid is used to rapidly redirect the air stream from the chamber toa route which bypasses the chamber. This creates a discreet pulse ofstimulant in the air stream. A second, outer Teflon© tube with adiameter of 2 mm surrounds the catheter-electrode assemblage, and itscentral end is connected to an aspirator that provides continuoussuction of 3 ml/s. This concentric arrangement of the outer suction tubeallows the emitted chemosensory stimuli to be localized to an area wecall a “minifield” (approx. dia=1 mm), and it avoids diffusion ofsubstances either to the area outside the intended stimulation site orinto the respiratory system. The entire stimulating and recordingassemblage may be positioned either on the neurosensory epitheliumwithin the VNo, or on the surface of the olfactory or respiratoryepithelium.

Electro-vomeronasogram (EVG)

Recordings are carried out in a quiet room with the subject supine; themulti-functional miniprobe is initially stabilized within the nasalcavity using a nasal retractor placed in the vestibule. Reference andground electrodes consist of silver discs (8 mm), both of which arepositioned on the glabella.

The entrance to the VNO, or vomeronasal pit, is identified by firstdilating the nasal aperture and vestibule. A 6×magnifying binocularloupe with halogen illumination is then used to introduce the tip of theTeflon© catheter and recording electrode assemblage into the VNo openingwhere it is stabilized at an approximate depth of 1 mm within thevomeronasal passage. Optimal placement of the recording electrode issignaled after testing for an adequate depolarization in response to atest substance.

Electrical signals from the recording electrode are fed to a DCamplifier after which they are digitized, computer monitored, andstored. The peak-to-peak amplitude of the signals is measured, and thearea under the depolarization wave is integrated, while continuouslymonitoring the signal both on the computer screen and on a digitaloscilloscope. Artifacts produced by respiratory movements are deleted bytraining the subjects to practice mouth breathing with velopharyngealclosure. Samples of vomeropherins in concentration of 25-800 fmoles aredelivered in the continuous air stream for durations from 300milliseconds to 1 second. Usually, intervals of 3 to 5 minutes separatedeach series of short test pulses. All components of the lines carryingthe test stimuli are made of Teflon©, glass or stainless steel and arecarefully cleaned and sterilized before each use. Activity was recordedusing standard electroencephalographic (EEG) electrodes placed atpositions Cz-Al and Tz-Al of the international 10120 system; the groundelectrode was placed on the mastoid process. Skin temperature (ST) wasrecorded by a small (1.0 mm) thermistor probe placed in the right earlobe. Respiratory frequency (RF) was measured with an adjustable straingauge placed around the lower thorax. All electrical signals were DCamplified, digitized (MP-100, Biopac systems) and continuously monitoredutilizing a computer.

Statistical Analysis

EVGs, peak-to-peak changes and frequency changes of other parameterswere measured and statistically analyzed. The significance of theresults was determined by either using paired t-tests or analysis ofvariance (ANOVA). Results on the EVG amplitude tested in the VNO of men(FIG. 1) and women (FIG. 2) are shown for steroids E2/NC2, E1/NC2,E2/NC3, E1/NC3, methylated E2/NC2, methylated E2/NC3, and E8/NC3.

Reflex Effects of Vomeropherins

Studies were conducted to determine the central nervous system (CNS)reflex responses to vomeropherin stimulation of the VNO. The sexuallydimorphic local responses induced by vomeropherins were sometimesmirrored in the autonomic response of male & female subjects.

Cortical activity was recorded from Cz and Tz in male and femalesubjects during application to the VNO of air pulses (300 ms to 1 sec)containing 200 fmoles of vomeropherin. There is also preliminaryevidence that the EVG is not associated with trigeminal nociceptorendings since application of a local anesthetic (2% lidocaine) to therespiratory epithelium of the nasal septum neither blocks nor diminishesthe EVG (Monti-Bloch, L. and Grosser, B. l. (1991) “Effect of putativepheromones on the electrical activity of the human vomeronasal organ andolfactory epithelium,”J. Steroid Biochem. Molec. Biol. 39:573-582.),also, subjects failed to report sensations of pain as a consequence ofany of the stimulation procedures.

What is claimed is:
 1. A compound selected from the group consisting of19-norchola-4,20-dien-3-one, 19-norchola-1,3,5(10),20-tetraen-3-ol,19-norchola-4,20-dien-3β-ol, 19-norchola-1,3,5(10),6,20-pentaen-3-ol,19-norchola-4,9,20-trien-3-one,19-nor-3-hydroxychola-1,3,5(10),20-tetraen-6-one,19-nor-10β-hydroxychola-4,20dien-3-one,19-nor-3-methoxychola-2,5(10),20-triene,19-norchola-1,3,5(10),7,20-pentaen-3-ol,19-norchola-1,3,5,7,9,20-hexaen-3-ol, 19-norchola-5(10),20-dien-3β-ol,19-norchola-1,3,5(10),20-tetraen-3,6β-diol,19-norchola-5(10),20-dien-3-one,19-norchola-1,3,5(10),9(11),20-pentaen-3-ol,19,21-bisnorchola-4,17(20)-dien-3-one,19,21-bisnorchola-4,17(20)-dien-3β-ol,19,21-bisnorchola-1,3,5(10),6,17(20)-pentaen-3-ol,19,21-bisnorchola-4,9,17(20)-trien-3-one,19,21-bisnor-3-hydroxychola-1,3,5(10),17(20)-tetraen-6-one,19,21-bisnor-10β-hydroxychola-4,17(20)-dien-3-one,19,21-bisnor-3-methoxychola-2,5(10),17(20)-triene,19,21-bisnorchola-1,3,5(10),7,17(20)-pentaen-3-ol,19,21-bisnorchola-1,3,5,7,9,17(20)-hexaen-3-ol,19,21-bisnorchola-5(10),17(20)-dien-3β-ol,19,21-bisnorchola-1,3,5(10),17(20)-tetraen-3,6β-diol,19,21-bisnorchola-5(10),17(20)-dien-3-one,19,21-bisnorchola-1,3,5(10),9(11),17(20)-pentaen-3-ol,19,21-bisnorchol-4-en-3-one, 19,21-bisnorchol-4-en-3β-ol,19,21-bisnorchola-1,3,5(10),6-tetraen-3-ol,19,21-bisnorchola-4,9-dien-3-one,19,21-bisnor-3-hydroxychola-1,3,5(10)-trien-6-one,19,21-bisnor-10β-hydroxychol-4-en-3-one,19,21-bisnor-3-methoxychola-2,5-(10)-diene,19,21-bisnorchola-1,3,5(10),7-tetraen-3-ol,19,21-bisnorchola-1,3,5,7,9-pentaen-3-ol,19,21-bisnorchol-5(10)-en-3β-ol,19,21-bisnorchola-1,3,5(10)-trien-3,6β-diol,19,21-bisnorchol-5(10)-en-3-one,19,21-bisnorchola-1,3,5(10),9(11)-tetraen-3-ol,19,21-bisnorchola-4,16-dien-3-one,19,21-bisncrchola-1,3,5(10),16-tetraen-3-ol,19,21-bisnorchola-4,16-dien-3β-ol,19,21-bisnorchola-1,3,5(10),6,16-pentaen-3-ol,19,21-bisnorchola-4,9,16-trien-3-one,19,21-bisnor-3-hydroxychola-1,3,5(10),16-tetraen-6-one,19,21-bisnor-10β-hydrocxychola-4,16-dien-3-one,19,21-bisnor-3-methoxychola-2,5(10),16-triene,19,21-bisnorchola-1,3,5(10),7,16-pentaen-3-ol,19,21-bisnorchola-1,3,5,7,9,16-hexaen-3-ol,19,21-bisnorchola-5(10),16-dien-3β-ol,19,21-bisnorchola-1,3,5(10),16-tetraen-3,6β-diol,19,21-bisnorchola-5(10),16-dien-3-one,19,21-bisnorchola-1,3,5(10),9(11),16-pentaen-3-ol,19,21-bisnorchola-4,16-dien-20(22)-yn-3-one,19,21-bisnorchola-1,3,5(10),16-tetraen-20(22)-yn-3-ol,19,21-bisnorchola-4,16-dien-20(22)-yn-3β-ol,19,21-bisnorchola-1,3,5(10),6,16-pentaen-20(22)-yn-3-ol,19,21-bisnorchola-4,9,16-trien-20(22)-yn-3-one,19,21-bisnor-3-hydroxychola-1,3,5(10),16-tetraen-20(22)-yn-6-one,19,21-bisnor-10β-hydroxychola-4,16-dien-20(22)-yn-3-one,19,21-bisnor-3-methoxychola-2,5(10),16-trien-20(22)-yne,19,21-bisnorchola-1,3,5(10),7,16-pentaen-20(22)-yn-3-ol,19,21-bisnorchola-1,3,5,7,9,16-hexaen-20(22)-yn-3-ol,19,21-bisnorchola-5(10),16-dien-20(22)-yn-3β-ol,19,21-bisnorchola-1,3,5(10),16-tetraen-20(22)-yn-3,6β-diol,19,21-bisnorchola-5(10),16-dien-20(22)-yn-3-one,19,21-bisnorchola-1,3,5(10),9(11),16-pentaen-20(22)-yn-3-ol, and19,21-bisnor-3-methoxychola-1,3,5(10),16-tetraene.
 2. A compoundaccording to claim 1 that is3-hydroxy-19,21-bisnorchola-1,3,5(10),16-tetraene.
 3. A compoundaccording to claim 1 that is 3-methoxy-19,21-bisnorchola-1,3,5(10),16-tetraene.
 4. A compound according to claim 1that is 3-hydroxy-19,21-bisnorchola-1,3,5(10),7,17(20)-pentaene.
 5. Acompound according to claim 1 that is3-hydroxy-19,21-bisnorchola-1,3,5(10),6,8(9),17(20)-hexaene.
 6. Acompound according to claim 1 that is3β-hydroxy-19,21-bisnorchola-4,17(20)-diene.
 7. A compound according toclaim 1 that is 3-hydroxy-19,21-bisnorchola-1,3,5(10),6,17(20)-tetraene.8. A compound according to claim 1 that is10-hydroxy-19,21-bisnorchola-4,16-dien-3-one.
 9. A compound according toclaim 1 that is 3-hydroxy-19,21-bisnorchola-5(10),16-diene.